In DNA-dependent-DNA-Polymerase amplification reactions such as. Polymerase Chain Reaction (PCR) and Real-Time quantitative PCR (qPCR), generation of non-specific amplification products may occur. Artifacts are derived from inappropriate hybridization products designated as primer dimers (Rychlik, 1995) and primer-probe dimers, resulting from weak interactions between primers or primers and probes such as TaqMan™ and molecular beacon probes. Undesired products form as a result of weak complementarity between the 3′ ends of a primer to bases in non-target oligonucleotide strands in the reaction mix. This enables annealing of the primer to the non-target strand, followed by initiation and elongation of the non-specific dimer by the thermo stable DNA polymerase and leads to an efficient amplification of this undesired byproduct.
In real-time PCR this problem worsens due to appearance of non-specific byproducts that mask the detection of low concentrations of the target sequence after 30 cycles (Watson, 1989) in case of complementarity of at least one nucleotide at the 3′ end, and after cycle 40 in case of no 3′ complementarity at all. This problem is magnified in multiplex PCR amplification reactions and qPCR due to the presence of several primer sets and probes in the reaction mix, leading to deteriorated sensitivity and quality of the reaction. High Resolution Melt (HRM) analysis, which requires prior amplification of the target sequence to high copy number, is especially sensitive to the purity of the sample. The presence of post-amplification artifacts such as primer dimers or non-specific byproducts can make HRM results difficult to interpret.
Ferrie et al. (1992) showed that under cold-start conditions, every possible combination of two different primers in a multiplex reaction would generate primer-dimers, irrespective of any primer complementarity.
Non-specific amplification products can be reduced by careful primer design, use of stringent PCR protocols (Don et. al., 1991) and use of ‘hot-start’ enzymes (Chou et al., 1992; D'Aquila et al., 1991; TaqMan™ PCR Reagent Kit Protocol, P. E. Applied Biosystems). However, hot-start enzymes do “leak” and apparently there are no absolute hot-start enzymes.
Several methods for the reduction of non-specific amplification products in PCR have been described: US Patent Application 2003/0175769 discloses the use of poly-hydroxy-aryl-poly-acid with 3-6 ortho-hydroxy acid moieties as an additive to a nucleic acid amplification reaction that can result in prevention of primer dimer formation. European Patent Application EP 0866071 and PCT Application WO 2006/112818 disclose the use of covalently modified nucleotides at or near the 3′ end of the primer for reduction of non-specific amplification products, in particular primer dimers.
European Patent Application EP 1201768 discloses methods and reagents to reduce non specific amplification involving the use of oligonucleotide primers in which at least one of the 3 nucleotides at the 3′ end of the primer is a modified nucleotide selected from the group consisting of 2′-O-amino-methyl-nucleotides, 2′-amino-methyl-nucleotides, 2′-fluoro-nucleotides, and arabinose nucleotides.
U.S. Pat. No. 7,205,129 and PCT Application WO 01/64952 disclose the use of a method for the reduction of artifacts during nucleic acids amplification based on the use of template-deficient oligonucleotides as primers. These template-deficient oligonucleotides comprise template-deficient nucleotides, preferably at or near the 5′ end. These template-deficient nucleotides, which may be modified nucleotides, derivatized nucleotides, nucleotide analogs or ribonucleotides, cannot serve as templates for nucleic acid synthesis, i.e., they prevent the synthesis of a nucleic acid strand complementary to a nucleic acid strand containing a template-deficient nucleotide at or beyond the site of the template-deficient nucleotide. In other words, the template-deficient oligonucleotide primers disclosed in U.S. Pat. No. 7,205,129 and WO 01/64952 cannot be fully replicated. U.S. Pat. No. 7,205,129 and WO 01/64952 teach away from the present invention by blocking DNA elongation at or beyond the site of a template-deficient nucleotide.
The standard situation where the primers do not undergo any modification delimits the detection sensitivity of assays that rely on DNA dependent DNA polymerase amplification, such as qPCR and HRM. The appearance of amplification products after cycle 30 is defined as a “twilight zone”. The detection is questionable and further examinations such as Tm (melting temperature) analysis and sequencing of the product are necessary.
Blocking the formation of non-specific template-independent products will prevent primer inactivation and diversion of the reaction towards the formation of non-informative byproducts. Elimination or any postponement in the appearance of non-specific template-independent products in DNA-dependent DNA-polymerase amplification reactions should increase the reaction robustness, specificity and sensitivity.
The existing methods for eliminating or reducing artifacts in nucleic acid amplification reactions involve chemical modifications or insertion of stretches (above 2 bases in a row) of RNA bases into primers, that require specific enzymatic or other, complicated, chemical reactions, or modifying nucleotides at the 5′ end of the primers in order to allow sufficient priming.
Thus, there is an unmet need for simpler, more effective, and more economical methods for improved nucleic acid amplification reactions and especially for real-time PCR and high resolution melt analysis applications with improved specificity and sensitivity.